Biomedical subjects
Publications and source records attributed to G G Wagner.
Laboratory reared Ixodes scapularis proved to be an efficient vector of Babesia odocoilei Emerson and Wright between white-tailed deer (Odocoileus virginianus). Transtadial survival of the babesia occurred between nymph and adult stages of the tick, and the adult stage transmitted the babesia.
Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.
Serum samples collected from 581 white-tailed deer (Odocoileus virginianus) from Texas and from 124 white-tailed deer from Oklahoma were tested by the indirect fluorescent antibody technique against Babesia odocoilei. Prevalence of seropositive reactors varied from site to site in both states. Prevalence rates were statistically ranked as high, intermediate or low. Deer less than 12-mo-old had a significantly lower prevalence than all other age classes.
Erythrocytes containing inositol hexaphosphate (IHP) were administered to mice. Mice were then challenged with Babesia microti. Mice receiving IHP carrier erythrocytes had significantly lower percentages of parasitaemias on days 3, 5 and 7 after infection. Carrier erythrocytes containing IHP have altered P50 oxygen values. Thus, carrier erythrocytes containing IHP may be useful in treating naive animals before transporting into areas endemic for babesiosis.
Bovine erythrocyte exposure to isometamidium chloride causes increased osmotic fragility. Control cells tolerated up to 1 mg/ml drug with no effects. Carrier erythrocytes were highly susceptible to drug, with increased osmotic fragility and decreased encapsulation potential of sucrose and inulin. Scanning electron micrographs of control and carrier erythrocytes exposed to drug revealed the formation of enkephalocytes with carrier erythrocytes. Control erythrocytes showed greater tolerance to the drug. Apparently, access of the drug to the interior of the erythrocyte membrane allows the drug to be more interactive with the membrane.
The effect of T-2 toxin on host resistance to acute and latent Babesia microti infections was evaluated in mice and was compared with the effects of the immunosuppressive drugs dexamethasone and cyclophosphamide. Mice with acute or latent B microti infection were treated with 2 mg of T-2 toxin/kg of body weight, 0.2 mg of dexamethasone/kg, or 30 mg of cyclophosphamide/kg daily for 5 days. Treatment with dexamethasone or cyclophosphamide caused significant (P less than 0.05) increases in Babesia parasitemia during acute infection and significantly (P less than 0.05) prolonged the duration of parasitemia during acute babesiosis. Treatment with T-2 toxin caused a transient significant (P less than 0.05) increase in Babesia parasitemia on day 10 after acute infection and numerical, though statistically nonsignificant, increases in the maximal level and duration of parasitemia during acute babesiosis. Significant (P less than 0.005) recrudescence of parasitemia was observed in the dexamethasone- and cyclophosphamide-treated mice with latent Babesia infection. Treatment with T-2 toxin did not cause recrudescence of parasitemia in mice with latent Babesia infection.
Pyriforms and ring forms of Babesia odocoilei were detected in thin blood smears obtained from a white-tailed deer killed by a hunter in Anderson County, Texas. Erythrocytes from the deer were cultured and the parasites maintained through 8 serial subcultures during 1 mo. The parasite was successfully established in culture using Medium 199 supplemented with either 20% deer serum or 40% normal adult bovine serum. The highest parasitemia observed was 30% and more than 4 parasites per erythrocyte were often observed. Cultured B. odocoilei remained infective for a susceptible white-tailed deer.
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Mouse blood infected with Babesia rodhaini and containing an equal volume of 4 M dimethyl sulfoxide was infective after storage at -196 degrees C for 8 years. The Babesia organisms were still able to cause lethal infections after prolonged low temperature storage.
The protective effect and anti-Anaplasma complement-fixing and agglutinating antibody responses induced in yearling steers by vaccination with an inactivated Anaplasma marginale vaccine were evaluated by challenge exposure. Eleven 12- to 14-month-old Hereford X Angus steers were randomly allotted into 6 principals and 5 controls. The principals were injected IM with 5 ml of vaccine on days 0 and 21. On day 70, the 11 steers were challenge exposed by IV inoculation of A marginale-infected blood. After a prolonged prepatent period, the vaccinated steers developed a significantly (P less than 0.01) lower A marginale parasitemia (8.0%) than did the nonvaccinated controls (26.3%). The persistence of a greater than or equal to 0.5% parasitemia was significantly (P less than 0.025) reduced from 34 days in the control to 21 days in the vaccinated group. The percentage reduction in PCV was significantly (P less than 0.025) less in the vaccinated group (34%) as compared with the controls (57%). Complement-fixing and agglutinating antibody responses induced by vaccination did not persist for more than 21 days after the 2nd vaccine injection and did not interfere with positive seroconversion resulting from challenge exposure.
A serological survey of cattle in Mali was carried out to determine the prevalence of antibody activity to Babesia bovis and B. bigemina. It was found that the level of B. bovis infection as indicated by antibody activities was too low to be of immediate concern. However, the serological prevalence of B. bigemina was high and this may indicate a potential disease problem. It was also found that when zebu and N'Dama cattle grazed together the N'Dama were twice as likely to have positive titres to Babesia as were the zebus.
Metabolism in the erythrocytes of normal mice and mice infected with Babesia microti has been monitored non-invasively by high resolution 13C NMR spectroscopy. The conversion of [U-13C]glucose to lactate in both normal and infected cells together with the effect of the trypanocidal drug, 4,4'-diamidinodiazoaminobenzene diaceturate, on glycolytic rates were monitored. These studies show that erythrocytes utilize [U-13C]glucose at a rate of 3 X 10(-12) mumol cell-1 min-1 at 35 degrees C while parasitized cells consume 2.9 X 10(-11) mumol cell-1 min-1 and produce lactate as the sole end-product. This rate decreases to 9 X 10(-12) mumol cell-1 min-1 on the addition of 0.75 mM drug.
Mononuclear effector cells from the peripheral blood of Babesia bovis-infected cattle responded during initial infection and destroyed Fc-bearing target cells. The activity of these cells was measured in an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay utilizing chicken erythrocytes coated with bovine anti-chicken erythrocyte antibody as targets. The activity of these effector cells was enhanced during the parasitemic crisis and returned to normal levels of activity following the resolution of parasitemia. This suggests that ADCC mechanisms may be involved in resolution of infection of cattle with B. bovis.
Yearling calves were pre-treated intravenously (IV) with a 0.20 mg kg-1 dose of Corynebacterium parvum and challenged after 30 days by IV inoculation of 3 X 10(9) Babesia bigemina. The relatively low 0.20 mg kg-1 dose of C. parvum enhanced resistance as indicated by lower mean Babesia parasitemias and less severe decreases in packed-cell volumes than in non-treated calves, but failed to stimulate a significant (P less than or equal to 0.05) level of protection against B. bigemina challenge.
A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.
Tetracycline was encapsulated in erythrocytes with a dialysis technique. The effect of drug concentration (0.2 mg/ml of erythrocytes) on encapsulation of [14C]sucrose and [3H]tetracycline was reduction of the tetracycline encapsulation, but not [14C]sucrose. Carrier erythrocytes containing tetracycline reinjected in calves were studied for their pharmacokinetic constants. The drug half-life was 6.7 hours, with an overall elimination constant of 0.104 hour-1.
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In vivo immunization, fusion, antibody detection, and cryopreservation procedures for monoclonal antibody production against antigens of Brucella abortus are described. Splenocytes from BALB/c mice immunized with irradiated B. abortus S2308 were fused with Sp2/O-Ag14 myeloma cells and 61 hybridomas secreting anti-Brucella antibodies were cloned. Hybridoma antibody synthesis was detected effectively and most efficiently by enzyme-linked immunosorbent assays. Antibodies from clones of hybridoma A23 reacted with S19 and S2308 whole bacterial cells, while hybridoma B49 reacted primarily with alkali--treated lipopolysaccharides of S19, S1119.3 and S2308. Cryopreservation of clones had no major effect on antibody synthesis. The application of monoclonal anti-Brucella antibodies in the differential diagnosis of bovine brucellosis is discussed.